The overarching goal of the proposed project is to address a critical but almost completely unexplored aspect of MutS function in DNA mismatch repair-the mechanism by which information is transmitted long-distance between its DNA binding and ATPase active sites to enable its function in DNA repair. Mismatch repair (MMR) corrects a broad range of post-replication errors in DNA, including base-base mismatches and insertion/deletion loops, and is therefore an important guardian of genomic and cellular integrity. Defects in this essential and highly conserved DNA repair pathway result in a high mutator phenotype and consequent carcinogenesis. In humans, MMR defects prominently manifest in hereditary Lynch syndrome (LS), which substantially raises the risk of colorectal and endometrial cancers, among others. MMR initiates with MutS protein recognizing a base-pairing error as it scans DNA and licensing MutL protein to begin the process of strand removal and re-synthesis. The InSiGHT database on LS mutations has well over two thousand unique gene variants of these two core MMR proteins, with 55% found in MSH2 and MSH6 human homologs of bacterial MutS. Up to a third of the MutS variants are due to small changes (single amino acid mutations, small in-frame insertion/deletions), and for many the mechanistic basis of the defect is unknown. It follows that investigation of these proteins has considerable significance and is indeed underway, from biochemical to clinical levels, to determine how MutS and MutL variants disrupt MMR and to define their pathogenicity in hereditary and spontaneous cancers. Our group has contributed to the field by investigating the kinetic mechanisms of T. aquaticus, S. cerevisiae and human MutS proteins thus far. Key to understanding how MutS works (or doesn't) is understanding how it finds and recognizes a mismatch and then interacts with MutL to initiate MMR, and how it utilizes ATP to perform these functions. We have developed a multi-component kinetic view of the MutS reaction, including its transactions with DNA and associated conformational dynamics, and its coupled ATPase activity. Significant findings from our transient kinetic analysis include: (a) a slow step followig initial weak interaction between MutS and DNA, in which concerted isomerization of both molecules leads to tight mismatch binding/recognition, as well as (b) rapid, binary switching of MutS conformations linked to ATP binding and hydrolysis, which is (c) stalled after mismatch recognition to drive formation of the ATP-bound MutS sliding clamp, which in turn stimulates MutL function. These rate-limiting events govern MutS actions during initiation of MMR. The proposed research is driven by lack of knowledge of how the two MutS activities, DNA binding and ATPase, are coupled to enable its function in MMR. We submit that detailed mechanistic understanding of T. aquaticus MutS makes it an excellent system for testing models of allosteric communication-in this case between the distant DNA binding and ATPase active sites. A novel approach for predicting allosteric signaling pathways, based on energy-weighted protein-nucleic acid structure networks, combined with molecular dynamics simulations, has been applied recently to nucleotide- and/or DNA-bound MutS complexes. In the resulting model, several amino acids identified as important nodes of communication are conserved through evolution, and their mutations in human homologs are found in the LS cancer database, providing additional impetus for study. We propose to experimentally test the new model of allostery by determining whether key conserved residues are indeed critical for information transfer between the two active sites on MutS. Since the questions posed here are about transient events in the reaction-how does DNA binding/release modulate ATP binding/hydrolysis/product release and vice versa-the use of rapid kinetics methods is essential to address them effectively. Having developed and validated numerous rapid-quench and stopped-flow assays to measure individual events in the MutS reaction, we can effectively assess whether T. aquaticus MutS mutants specifically exhibit defects in allostery-dependent steps in the reaction. Complementary analysis of human MutS mutants will help assess the biological relevance of the model, and contribute important new information toward understanding Lynch syndrome. Our research group is the only one thus far to use ensemble transient kinetic analysis to determine detailed mechanisms of the core MMR proteins. Therefore, the proposed study is expected to contribute significant new information to the field of DNA mismatch repair. On a broader scale, the proposed experiments promote application of transient kinetic methods for analysis of complex systems comprising multiple macromolecules and small ligands. This push for high-resolution kinetic data nicely complements the push for high-resolution structural data on multi-component systems. Moreover, the rate and equilibrium parameters obtained from bulk kinetics are essential to inform interpretation of kinetic data from single molecule experiments. The research will be conducted by collaborative groups of graduate and undergraduate students who will be trained in fundamental principles and techniques of biochemistry-from the preparation of mutant recombinant proteins to thermodynamic and kinetic analysis of their interactions and catalytic activities, including pre-steady state kinetic methods. A complementary training goal of the project is to prepare graduating students to succeed at independent scientist and/or mentor positions in industry or academia.